Inhaled Combination Therapy

ABSTRACT

There is provided the use of a methylxanthine derivative such as theophylline and a steroid in a synergistic combination for the treatment of chronic obstructive pulmonary disease, wherein the combination is administered by the inhaled route for pulmonary delivery.

The present invention provides the inhaled use of methylxanthinederivatives such as theophylline and steroid drugs in a synergisticcombination for manufacture of a medicament in the treatment of chronicrespiratory diseases such as chronic obstructive pulmonary disease(COPD). The administration of a methylxanthine and a steroid incombination administered via the inhaled route for pulmonary delivery,results in a therapeutic synergistic pulmonary anti-inflammatoryresponse.

INTRODUCTION

Theophylline is an inexpensive white crystalline powder used as an oralagent for chronic respiratory diseases such as asthma and COPD.Aminophylline, or theophylline ethylenediamine, is a combination oftheophylline and ethylenediamine and has similar properties.Theophylline is known to have a bronchodilating effect and a mildanti-inflammatory effect, due in part to its activity as a weaknonselective phosphodiesterase (PDE) inhibitor. The drug has hithertobeen characterised by a narrow therapeutic index, and toxicity to thisagent, marked by gastrointestinal upset, tremor, cardiac arrhythmias,and other complications, is common in clinical practice. Other drugs forchronic respiratory diseases, such as inhaled beta-agonists, inhaledanti-cholinergics and inhaled steroids, are often prescribed instead oforal theophylline to avoid its adverse effects. However, to date therehas been limited investigation of the therapeutic potential of inhaledtheophylline in the treatment of chronic respiratory diseases such asCOPD and asthma.

Although theophylline has been in clinical use for many years, itsmolecular mechanism of action and its site of action remain uncertain.Several molecular mechanisms of action have been proposed, including thefollowing.

Theophylline is a weak and nonselective inhibitor of phosphodiesterases,which break down cyclic nucleotides in the cell, thereby leading to anincrease in intracellular cyclic AMP and GMP concentrations.Theophylline relaxes airway smooth muscle by inhibition of PDE activity(PDE3, PDE4 and PDE5), but relatively high concentrations are needed formaximal relaxation (Rabe, et al. Eur Respir J 1999, 8:637-42). Thedegree of PDE inhibition is very small at concentrations of theophyllinethat are therapeutically relevant. There is no evidence thattheophylline has any selectivity for any particular isoenzyme, such as,for example, PDE4B, the predominant PDE isoenzyme in inflammatory cellsthat mediates anti-inflammatory effects in the airways.

Theophylline increases interleukin-10 release, which has a broadspectrum of anti-inflammatory effects. This effect may be mediated viaPDE inhibition, although this has not been seen at the doses that areeffective in asthma (Oliver, et al. Allergy 2001, 56: 1087-90).

Theophylline prevents the translocation of the proinflammatorytranscription factor nuclear factor-κb (NF-κB) into the nucleus, thuspotentially reducing the expression of inflammatory genes in asthma andCOPD (Tomita, et al. Arch Pharmacol 1999, 359: 249-55). These effectsare seen at high concentrations and may also be mediated by inhibitionof PDE. It is likely therefore, that PDE inhibition is not a mechanismof action of theophylline when administered at low oral doses (<10mg/L).

Theophylline is also a potent inhibitor of adenosine receptors attherapeutic concentrations, with antagonism of A₁ and A₂ receptors,although it is less effective against A₃ receptors (Pauwels & Joos, ArchInt Pharmacodyn Ther 1995, 329: 151-60). There is a report that inhaledtheophylline monotherapy can antagonise the bronchoconstrictor activityof inhaled adenosine in asthmatics (Cushley et al, Am Rev Resp Disease1984, 129,380-4). In addition, inhaled theophylline (given asaminophylline) has also been shown to act as bronchodilator in asthmaticpatients (Kokubu H. Arerugi. 1995, 44(12): 1379-86).

Theophylline has moreover recently been shown to activate histonedeacetylase (HDAC). Acetylation of histone proteins is associated withactivation of gene function, and it is believed that proinflammatorytranscription factors which activate inflammatory genes also cause anincrease in histone acetyltransferase activity. By increasing HDACactivity and so deacetylating histone proteins, theophylline is believedto suppress the expression of inflammatory genes (see Barnes P J, Am JRespir Crit Care Med, 2003, 167: 813-818).

Theophylline is believed to interact with a key regulatory kinase orphosphatase enzyme in the molecular pathway which results in elevatedHDAC activity, although this is yet to be elucidated.

Minimal but significant pulmonary anti-inflammatory activity has beendemonstrated by oral theophylline monotherapy in COPD patients usingdoses of theophylline that gave plasma levels ≦10 mg/L but ≧5 mg/L(Culpitt et al. Am. J. Respir. Crit Care Med, 2002, 165(10): 1371-1376,Kobayashi et al, Respirology 2004, 9: 249-254).

Glucocorticoid drugs (steroids) have become the therapy of choice inasthma and are widely used in the treatment of COPD, usually in inhaledform. Inhaled steroids are usually administered once or twice daily at afixed dosage dependent on the properties of the steroid administered.For example, Budesonide is typically administered at doses of 200 mcg,400 mcg or 800 mcg doses once or twice daily. Fluticasone is typicallyadministered at doses of 50 mcg, 100 mcg, 250 mcg or 500 mcg doses onceor twice daily. Ciclesonide is typically administered at doses of 80mcg, 160 mcg or 320 mcg doses once or twice daily. The absolutepulmonary delivered dose is dependent on many factors (includingproperties of the steroid used, inhalation device characteristics,inhalant formulation, patient characteristics, and inhalation technique)but is normally significantly less than the total administered.

However, although inhaled steroids are effective in the majority ofasthma patients their use in COPD is contentious owing to their lack ofdemonstrable anti-inflammatory effect in sputum samples (Keatings et al.Am. J Respir. Crit Care Med, 1997, 155(2):542-8, Culpitt et al. Am. JRespir. Crit Care Med. 1999, 160, 5(1): 1635-1639, Loppow et al. Respir.Med. 2001, 95(2): 115-121) and their apparent failure to affect diseaseprogression (Burge, et al (2000). BMJ 320: 1297-1303). Other studiesexamining sputum have suggested that longer treatment periodsparticularly with high dose inhaled steroids do have measurable butminimal pulmonary anti-inflammatory effects in some patients(Confalonieri et al. Thorax, 1998, 53(7): 583-585, Mirici et al.Clinical Drug Investigation. 2001, 21: 835-842, Yildiz et al.Respiration. 2000, 67: 71-76).

Assessment of anti-inflammatory activity by examining bronchial biopsiesrather than sputum from COPD patients has also been disappointinghowever. In contrast to that demonstrated in asthma patients, Hattatowaet al. (Am J Respir Crit Care Med. 2002, 165: 1592-1596) did not detectsignificant reductions in the key inflammatory cells in COPD following 3months treatment with an inhaled steroid (500 mcg Fluticasoneadministered twice daily). Despite minimal pulmonary anti-inflammatoryactivity overall, ICS do reduce exacerbation frequency in COPD patientsby 20-30%.

It is likely that improving this minimal pulmonary anti-inflammatoryactivity of inhaled steroids would provide a great benefit to COPDpatients resulting in improved health status, improved pulmonaryfunction, improvement in measures of dyspnea and further reducedexacerbation frequency.

Asthma patients who fail to respond to low doses of steroids areadministered a higher dose, in the case of budesonide up to 1600 mcgdaily. Evans et al, (2004) NEJM 337:1412, suggest that high doses ofinhaled steroids may be substituted by administration of a normalglucocorticoid dose, together with a low dose of oral theophylline foruse in asthma. Patients were administered 400 mcg of budesonide (thestandard dose) together with 250 or 375 mg of theophylline, or 800 mcgof budesonide plus placebo, twice daily. The plasma concentrations oftheophylline that were achieved in this study ranged from 2.5 to 17.1mg/l with a median value of 8.7 mg/l. The effects of these two treatmentparadigms were similar suggesting that theophylline has dose sparingeffects when given with a steroid. However, at the doses used, patientssuffered from drug-related side effects, including gastrointestinalupsets, palpitations, sore throats and other side-effects associatedwith steroids and/or theophylline therapy. The authors did not determineany effects of the drug combination on inflammation. Similar studiesinvestigating potential dose sparing of inhaled steroids with oraltheophylline have not been carried out in COPD patients.

Cazzola et al, (2004) Pulm Pharm Ther 17:141-145, compared thecombination of the bronchodilator salmeterol and the steroid fluticasonewith the combination of oral theophylline and inhaled fluticasone (500mcg twice daily) in COPD patients over a period of 4 months. The dose oforal theophylline was titrated to achieve plasma concentrations of 10-20mg/L as these plasma levels are required for clinical efficacy in termsof bronchodilation. The inflammatory status of these patients was notexamined with either therapy.

US20030134865 discloses a screening method for identifying drug-likecompound for treating asthma or other inflammatory diseases. Itcomprises of a xanthine or a related compound to modulate the activityor expression of HDAC. The invention describes the role of steroids inthe repression of inflammatory genes. It predicts that theophyllineshould enhance glucocorticoid actions by enabling glucocorticoids torecruit HDACs with increased activity. Theophylline is used insynergistic effect with the steroids Dexamethasone and Budesonide inenhancing cell HDAC activity and repression of inflammatory genes invitro.

U.S. Pat. No. 6,025,360 discloses the use of theophylline as an add-ontherapy or to replace completely or partially treatment withcorticosteroids for chronic, prophylactic, anti-inflammatory treatmentof asthma to provide a mean steady state plasma level of 1 to less than5 mg/L. The plasma levels are achieved by preparing theophyllineadministrated by the oral route in a dosage form for dosing at 50 to 600mg/day.

Barnes (Am J Respir Crit Care Med, 2003, 167:813-818), discusses the useof theophylline and steroids in combination for COPD and suggests thatthe combination may have a synergistic effect in reducing inflammation.However, no dosing regime is provided nor is the use of administratingtheophylline by the inhaled route disclosed.

For clarity Table 1 presents a summary of the prior art in regard tooral doses of methylxanthines administrated and correspondingtheophylline plasma levels detected in patients.

For clarity Table 2 presents a summary of the limited data in the priorart in regard to inhaled doses of methylxanthines administrated andcorresponding theophylline plasma levels detected in asthma patients.

TABLE 1 Average Study theophylline Publication duration Drug Daily doseplasma level Alexander et al. (1980) J 4 weeks Theophylline 400 mg 15.1mg/L  Am Med Assoc. 244: 2286-90 Iversen et al. (1992) Eur 4 weeksTheophylline 600 mg 7.1 mg/L Respir J. 5 Suppl (15): 137. Ito et al.(2002). 4 weeks Euphylong 500 mg 4.3 mg/L Proc. Natl. Acad. Sci. USA(Theophylline) 99(13): 8921-8926 Culpitt et al. (2002) 4 weeksTheophylline 300-600 mg 9.5 mg/L Am. J. Respir. Crit Care Med. 165(10):1371-1376 Kobayashi et al (2004). 4 weeks Theodur 400 mg 7.9 mg/LRespirology 9: 249-254 (Theophylline) Evans et al., (2004) NEJM 12weeks  Euphylong 500-750 mg 8.7 mg/L 337: 1412 (Theophylline) U.S. Pat.No. 6,025,360 4 weeks Phyllocontin or 562-573 mg Average not quoted(1995) Uniphylline but 20/25 patients (Theophylline) had levels ≧5 mg/L

TABLE 2 Net peak Study theophylline Publication duration Drug Doseplasma level Bohadana et al. Single Aminophylline 187.5 mg 0.91 mg/L(1980) Bull. Eur. inhaled (Theophylline) Physiopathol. exposure Respir.16(l): 13-24.

Thus, there is a medical need for a therapeutic regime for COPD whichprovides effective anti-inflammatory activity and avoids side-effectsassociated with existing therapies. The combination of inhaledtheophylline with an inhaled steroid in the manufacture of a medicamentis disclosed in our copending international patent applicationPCT/GB05/003039 but has not been described in the published literature.Such a medicament provides a combination therapy for COPD and otherchronic respiratory diseases that may improve the pulmonaryanti-inflammatory activity of the inhaled steroid. This inhaledcombination produces minimal methylxanthine systemic exposure (plasmalevels <1 mg/L) and therefore results in an attractive side effectprofile compared with chronic oral administration.

BRIEF DESCRIPTION OF THE INVENTION

The present inventors have determined that steroids administeredtogether with methylxanthine compounds by the inhaled route at doseswhich alone are not effective in treating inflammation induced bytobacco smoke (TS) in an animal model of COPD, have a synergistic effectand are able to markedly reduce inflammation in said models, for exampleby 44% or more in the tests set forth below. TS exposure is widelyaccepted to be the principal cause of COPD in human beings.

In a first aspect, therefore, there is provided the use of amethylxanthine compound and a steroid in the manufacture of acomposition for the treatment of a chronic respiratory disease selectedfrom the group consisting of chronic obstructive pulmonary disease(COPD), cystic fibrosis, steroid resistant asthma, severe asthma andpaediatric asthma in a human subject, wherein the composition isadministered by the inhaled route for pulmonary delivery, and themethylxanthine compound is administered at a dose between 0.1 and 25mg/day.

Advantageously, the inhaled steroid is administered at a dose which, inisolation, is not effective in reducing the inflammation associated withthe said respiratory disease. The inhaled steroid may also beadministered at a dose which, in isolation, has minimal efficacy withrespect to improvements in lung function and inflammation in treatingsaid respiratory disease.

There is also provided the use of the inhaled combination wherein theinhaled steroid is administered at a lower dose than that, in isolation,has significant efficacy with respect to improvements in lung functionand inflammation in treating said respiratory disease (steroid sparing).

Improving this minimal pulmonary anti-inflammatory activity of inhaledsteroids (such as budesonide, fluticasone or ciclesonide) using aninhaled methylxanthine (such as theophylline) in combination provides agreat benefit to patients suffering from chronic respiratory diseasessuch as COPD by resulting in improved health status, improved pulmonaryfunction, improvement in measures of dyspnea and further reducedexacerbation frequency.

The invention recognises a synergistic activity between a methylxanthinecompound and steroid drugs which results in an extremely high pulmonaryanti-inflammatory activity. This synergy can be achieved using doses ofthe drugs which were ineffective when administered alone. The effect isnot additive, but synergistic, in that two drugs having little or noeffect can be administered simultaneously to obtain highly significantinhibition of the inflammatory response.

A methylxanthine compound, as used herein, refers to theophylline andpharmacologically equivalent compounds and salts, includingaminophylline and oxtriphylline. Such compounds are methylxanthines,which include caffeine, acepifylline, bamifylline, bufylline, cafaminol,cafedrine, diprophylline, dihydroxypropyltheophylline, doxofylline,enprofylline, etamiphylline, etofylline, proxyphylline, suxamidofylline,Theobromine, Furaphylline, 7-propyl-theophyllinedopamine,Pentoxifylline, Reproterol, denbufylline, IBMX, Arofylline,Cipamfylline, HWA 448, SDZ MKS 492, BB-1502 and the like.

Steroid drugs include glucocorticoids, corticosteroids andmineralocorticoids, such as dexamethasone and budesonide,beclomethasone, beclomethasone dipropionate, flunisolide, fluticasone,Ciclesonide, mometasone, hydrocortisone, prednisone, prednisolone,methyl prednisolone, naflocort, deflazacort, halopredone acetate,fluocinolone acetonide, fluocinonide, clocortolone, tipredane,prednicarbate, alclometasone dipropionate, halometasone, rimexolone,deprodone propionate, triamcinolone, betamethasone, fludrocoritisone,desoxycorticosterone, rofleponide, etiprendnol dicloacetate and thelike. Steroid drugs can additionally include steroids in clinical orpre-clinical development for respiratory diseases such as GW-685698,GW-799943, NCX-1010, NCX-1020, NO-dexamethasone, PL-2146, NS-126(formerly ST-126) and compounds referred to in international patentapplications WO0212265, WO0212266, WO02100879, WO03062259, WO03048181and WO03042229. Steroid drugs can also additionally include nextgeneration molecules in development with reduced side effect profilessuch as selective glucocorticoid receptor agonists (SEGRAs), includingZK-216348 and compounds referred to in international patent applicationsWO-00032585, WO-000210143, WO-2005034939, WO-2005003098,WO-2005035518and WO-2005035502.

Preferably, the methylxanthine is theophylline.

Preferably, the steroid is budesonide, fluticasone or ciclesonide.

In accordance with the invention, the steroid may be administered at astandard dose, or a dose which would have a minimal effect ifadministered independently of the methylxanthine compound to anindividual.

Advantageously, the steroid is ineffective in reducing inflammation insaid respiratory disease at the dose used. Certain respiratory diseases,including COPD, are resistant to steroid treatment and steroid drugs areminimally effective in reducing pulmonary inflammation. Together with amethylxanthine compound such as theophylline, however, a pulmonaryanti-inflammatory effect is observed.

Administration takes place via the inhaled route, providing theconvenience of patients receiving their target doses of an inhaledmethylxanthine compound and inhaled steroid, with or without a longacting bronchodilator drug, from a single device. Typically the longacting bronchodilator drug would be either a long-acting β-agonist, suchas salmeterol or formoterol, or a long acting anti-muscarinic, such astiotropium.

Administration of the steroid may also be by the oral route with themethylxanthine compound being given by inhalation.

The combinations of the invention can optionally comprise one or moreadditional active substances which are known to be useful in thetreatment of respiratory disorders such as PDE4 inhibitors, PDE5inhibitors, PDE7 inhibitors, leukotriene D4 inhibitors, leukotriene B4inhibitors, inhibitors of egfr-kinase, p38 MAP kinase inhibitors, NF-κBpathway inhibitors such as IκK inhibitors, A2A adenosine receptoragonists, TNFα signalling inhibitors (such as ligand binding agents,receptor antagonists), Interleukin-1 signalling inhibitors, CRTH2receptor antagonists, protease inhibitors (such as neutrophil elastaseinhibitors, MMP inhibitors, Cathepsin inhibitors), IL-8 signallingmolecules, CXCR1inhibitors, CXCR2 inhibitors, iNOS modulators, PPARagonists, anti-oxidants (including N-acetylcysteine and superoxidedismutase mimetics) and/or NK-1 receptor antagonists.

In another aspect, the invention provides a pharmaceutical compositioncomprising a methylxanthine compound and a steroid for use in thetreatment of a chronic respiratory disease selected from the groupconsisting of chronic obstructive pulmonary disease (COPD), cysticfibrosis, steroid resistant asthma, severe asthma and paediatric asthma,wherein the composition is adapted for administration by the inhaledroute for pulmonary delivery and wherein the methylxanthine compound isadministered at a dose of 0.1 to 25 mg/day.

Moreover, the invention provides a methylxanthine compound and a steroidfor simultaneous, simultaneous separate or sequential use in thetreatment of a chronic respiratory disease selected from the groupconsisting of chronic obstructive pulmonary disease (COPD), cysticfibrosis, steroid resistant asthma, severe asthma and paediatric asthma,wherein the composition is adapted for administration by the inhaledroute for pulmonary delivery and wherein the methylxanthine compound isadministered at a dose of 0.1 to 25 mg/day.

Preferably, the methylxanthine compound is provided in an amount from0.1 and 25 mg, or from 0.05 to 13 mg, or from 0.033 to 9 mg per unitdose of the composition, said unit doses corresponding respectively tothose for once per day, twice per day, or three times per day treatment.Preferably, the daily dose is between 1 and 24 mg, 23 mg, 22 mg, 21 mgor 20 mg. For example, the daily dose may be 5, 10, 15, 16, 17, 18 or 19mg. Unit dosages may be calculated for once, twice or three times dailyapplication.

Preferably, the methylxanthine compound is theophylline. Advantageously,the theophylline is administered at a dosage as set forth above.

Methylxanthines other than theophylline may be administered in the abovedosages, or at dosages which may be adapted from the above by personsskilled in the art.

The steroid may be provided, in general, at a dosage of from about 40mcg to 1600 mcg, either in a single daily does or in multiple unit doseswhich together add up to the stated dose. For example, the steroid maybe provided in a daily dose of 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 750, 800, 900 or 1000 mcg.

Advantageously, the steroid is budesonide and is provided in an amountfrom 200 mcg to 1600 mcg, or from 100 mcg to 800 mcg mg, or from 65 mcgto 525 mcg per unit dose of the composition, said unit dosescorresponding respectively to those for once per day, twice per day, orthree times per day treatment. Preferably, the daily dose is between 200mcg and 1600, 1500, 1400, 1300, 1200, 1100 or 1000 mcg; for example, thedaily dose is 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850 or 900 mcg. Unit dosages may be calculated for once, twice orthree times daily application.

In a further embodiment the steroid is fluticasone and is provided in anamount from 44 mcg and 1000 mcg, or from 22 mcg to 500 mcg, or from 15mcg to 335 mcg per unit dose of the composition, said unit dosescorresponding respectively to those for once per day, twice per day, orthree times per day treatment. Preferably, the daily dose is between 44mcg and 900, 880, 800, 700, 660, 600 or 500 mcg; for example, the dosecan be 50, 60, 88, 100, 110, 125, 150, 200, 220, 250, 300, 350, 375,400, 440 or 450 mcg. Unit dosages may be calculated for once, twice orthree times daily application.

Moreover, the steroid can be Ciclesonide and be provided in an amountfrom 40 mcg and 500 mcg, or from 20 mcg to 250 mcg mg, or from 17 mcg to170 mcg per unit dose of the composition, said unit doses correspondingrespectively to those for once per day, twice per day, or three timesper day treatment. The daily dose is preferably between 40 mcg and 480,450, 400, 350, 320 or 300 mcg; for example, the dose is 80, 100, 120,150, 160, 200 or 250 mcg. Unit dosages may be calculated for once, twiceor three times daily application.

In one embodiment, the corticosteroid is mometasone furoate and isprovided in an amount from 50 μg to 1000 μg, or from 110 μg to 880 μg orfrom 200 μg to 500 μg per unit dose of the composition, said unit dosescorresponding respectively to those for once per day, twice per day, orthree times per day treatment. Preferably, the daily dose is between 50μg and 900, 850, 800, 750, 700, 650 or 600 μg: for example, the dailydose is 55, 110, 220, 440, 660, or 880 μg. Unit dosages may becalculated for once, twice or three times daily application. Mometasonefuroate is commercially available as an inhalation powder from Merck(ASMANEX®).

In another embodiment, the corticosteroid is beclomethasone dipropionate(also known as beclometasone dipropionate) and is provided in an amountfrom 20 μg to 2000 μg or from 40 μg to 800 μg per unit dose of thecomposition, said unit doses corresponding respectively to those foronce per day, twice per day, or three times per day treatment. Forexample, the daily dose is 40, 50, 80, 100, 120, 150, 160, 200, 240,250, 280, 300, 320, 350, 360, 400, 500, 600, 640, 800. Unit dosages maybe calculated for once, twice or three times daily application.Beclomethasone dipropionate is commercially available from Teva BrandedPharma (QVAR 40 and QVAR 80).

The corticosteroids mometasone furoate or beclomethasone dipropionatemay also be administered in the form of combinations with long actingbronchodilators with a range of mechanisms including beta 2 adrenergicagonists (such as formoterol or salmeterol) and/or muscarinicantagonists such as ipratropium and tiotropium. For example formoterolfumarate and beclomethasone dipropionate or formoterol fumarate andmometasone furoate. The bronchodilator included in the steroidcombination can have beta 2 adrenergic agonist and muscarinic antagonistactivity in the same molecule.

Such unit dosages may be packaged to provide a kit for the treatment ofsaid respiratory disease, comprising a methylxanthine compound and asteroid in unit dosage form, wherein the composition is administered bythe inhaled route for pulmonary delivery. For example, a kit accordingto the invention may comprise the methylxanthine compound and thesteroid in admixture, in the correct ratio, for concurrentadministration. Alternatively, the compounds may be provided as separatecompositions for simultaneous separate or sequential administration.

In a particularly preferred embodiment, the methylxanthine compound andthe steroid are provided in the form of an inhaler, for administrationvia the inhaled route. The inhaler may be a single bottle inhaler, withthe compounds provided in admixture, or a twin-bottle inhaler whereinthe compounds are provided separately but administered together.

Such a kit may comprise, for example, instructions for use which directthe user to administer the medicaments substantially simultaneously,such that they are present in the patient's body, particularly thelungs, at the same time.

In the kits or unit dosages according to the invention, the steroid ispreferably present at a dose which is insufficient to be effective inthe treatment of said respiratory disease if administered independently.

Importantly, plasma levels achieved with the doses of a methylxanthinecompound such as theophylline used (total daily administered dose 0.1-25mg) fall below those currently considered necessary for clinicalefficacy (10-20 mg/l) (Cazzola et al, Pulmonary Pharmacology &Therapeutics, 2004, 17, 141-145) and those below used to demonstrateanti-inflammatory activity (5 mg/L) (Culpitt et al. Am. J. Respir. CritCare Med, 2002, 165(10): 1371-1376, Kobayashi et al, Respirology, 2004,9:249-254). The nominal dose quoted as being delivered may exceed thisfigure, depending upon the characteristics of the device and theformulation in use; however, the invention provides that an effectivedose resulting in plasma levels between 1 pg/L and 1 mg/L ofmethylxanthine determined 1 hour post administration is effective intreating respiratory conditions when administered by the inhaled routefor pulmonary delivery in combination with a steroid drug.

Preferably, the plasma level of methylxanthine compound achieved asdescribed above is between 1 mcg/L and 1 mg/L, advantageously 100mcg/L-1 mg/L, 250 mcg/L-1 mg/L, 500 mcg/L-1 mg/L or 750 mcg/L-1 mg/L.Advantageously, it is 950 mcg/L or less, 900 mcg/L or less, 850 mcg/L orless, or 800 mcg/L or less.

In the foregoing aspects of the invention, the dosage of steroid whichdoes not exert any apparent pharmacological effect in the animal modelof COPD is advantageously below 0.5 mg/kg, preferably between 0.1 and0.4 mg/kg, most preferably between 0.1 and 0.3 mg/kg. Advantageously,the dose of steroid is 0.1 mg/kg or less.

There is moreover provided a method for the treatment of a chronicrespiratory disease selected from the group consisting of chronicobstructive pulmonary disease (COPD), cystic fibrosis, steroid resistantasthma, severe asthma and paediatric asthma, comprising administering toa human patient in need thereof a composition according to the foregoingaspects of the invention.

The effectiveness of the treatment may be assayed, in accordance withthe invention, by any technique capable of assessing inflammation. In apreferred embodiment, the treatment of the respiratory disease isassessed by counting cells recovered by bronchoalveolar lavage (BAL).Inflammation can also be assessed in sputum or in bronchial epithelialbiopsies (Rutgers et al. Eur Respir J. 2000, 15(1):109-15). Non-invasiveassessment of pulmonary inflammation can also be assessed using positronemission tomography and ¹⁸F-fluorodeoxyglucose uptake (Jones et al. EurRespir J, 2003, 21: 567-573).

Advantageously, the pulmonary cells are selected from the groupconsisting of macrophages, neutrophils and lymphocytes.

The invention is capable of substantially reducing inflammation inrespiratory diseases. Advantageously the cell count is reduced by 40% ormore upon administration of a methylxanthine compound and a steroid,preferably 44%, 50%, 60% or more.

At the same time, the individual doses of a methylxanthine compound andthe steroid can advantageously reduce cell numbers by a total, whenadded together, of 31% or less, preferably 30% or less, and ideally by20% or less. Where the synergistic reduction of cell count onadministration of a methylxanthine compound and a steroid is 40% ormore, preferably 44% and advantageously 50% or more, the additive effectof the individual agents is preferably 31% or less, advantageously 20%or less.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Effect of Theophylline, Dexamethasone or their combination,administered orally or intra-nasally, on total cell numbers recovered inthe BAL 24 hr post final exposure.

FIG. 2 Plasma levels of Theophylline at 1 hr post administration whenadministered in combination with Dexamethasone by either the oral orintranasal route in TS exposed mice.

FIG. 3 Effect of Theophylline, Dexamethasone or their combination,administered orally or intra-nasally, on macrophage numbers recovered inthe BAL 24 hr post final exposure.

FIG. 4 Effect of Theophylline, Dexamethasone or their combination,administered orally or intra-nasally, on neutrophil numbers recovered inthe BAL 24 hr post final exposure.

FIG. 5 Effect of Theophylline, Dexamethasone or their combination,administered orally or intra-nasally, on lymphocyte numbers recovered inthe BAL 24 hr post final exposure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs standard techniques of pharmacology andbiochemistry, as described in more detail below. In the context of theinvention, certain terms have specific meanings, as follows.

The invention describes the administration of theophylline and steroiddrugs in combination, and contrasts the combined administration withindividual administration of said drugs in isolation. “In isolation”accordingly refers to the administration of theophylline without asteroid, or vice versa, irrespective of whether the steroid isadministered before, concomitantly with or after the theophylline. Theintention is to differentiate between the theophylline and the steroidbeing administered such that they can exert their pharmacologicalactivities in the target organism contemporaneously or separately.

“Combined use” or “combination” within the meaning of the presentinvention is to be understood as meaning that the individual componentscan be administered simultaneously (in the form of a combinationmedicament), separately but substantially simultaneously (for example inseparate doses) or sequentially (directly in succession or after asuitable time interval, provided that both agents are active in thesubject at the same time).

“Effective”, referring to treatment of inflammatory conditions and/orrespiratory disease, refers to obtaining a response in an assay whichmeasures inflammation in respiratory disease. The preferred assay isbronchoalveolar lavage (BAL) followed by cell counting, wherein thepresence of cells indicates inflammation of the lung. In human patients,BAL, induced sputum and bronchial biopsy are preferred methods ofassessing inflammation. Inflammation may be induced by any desiredmeans, such as tobacco smoke inhalation, administration of irritantssuch as LPS, and the like. Tobacco smoke inhalation is preferred since,as shown previously in UK patent application 0417481.9, the use of LPSdoes not faithfully reproduce an inflammatory response that is steroidresistant as is seen in COPD. In the context of the BAL/cell countingassay, “effective” preferably encompasses a reduction in cell numbers by30%, 35%, 40%, 45%, 50%, 60%, 65%, 70% or more compared to a control inwhich the agent is not administered.

“Not effective” means, in the same assay, a much lower level ofresponse. Preferably, in the BAL/cell counting assay, “not effective”means that the reduction in cell numbers is 30% or below, advantageously28%, 27%, 26%, 25%, 24%, 23%22% or 21% or below, and preferably 20% orbelow. In some instances, “not effective” can encompass an increase ininflammation, seen for example as an increase in cell numbers.

“Synergistic” means that the effectiveness of two agents is more thanwould be expected by summing their respective individual effectivenessin a given assay. For example, if theophylline and a steroid reduce cellnumbers in the BAL assay by 10% and 20%respectively when administered inisolation, a synergistic response would be seen if the reduction in cellnumbers were above 30%, preferably above 35%, 40%, 50% or more, in acombined administration of the same agents at the same dose.

“Administered” refers to the administration of the entire dose of theagent, such as in a bolus dose, to the intended subject. In the contextof the present invention, dosage is preferably expressed in terms ofplasma levels achieved (<5 mg/L; 6-9 mg/L; 10-20 mg/L) with plasmalevels preferably less than 5 mg/L, and most preferably less than 1mg/L.

A “dose” is an amount of agent administered as described above.

“Unit dosage” form is a preparation of a pharmaceutical composition inone or more packaged amounts, each of which contains a single dosage inaccordance with the invention.

Methylxanthines and Steroids

Theophylline and Aminophylline

Theophylline has the structure shown below:

and is available commercially under a variety of brand names, includingAccurbron, Aerobin, Aerolate, Afonilum, Aquaphyllin, Armophylline,Asmalix, Austyn, Bilordyl, Bronchoretard, Bronkodyl, Cetraphylline,Constant T, Duraphyllin, Diffumal, Elixomin, Elixophyllin, Etheophyl,Euphyllin, Euphylong, LaBID, Lanophyllin, Lasma, Nuelin, Physpan,Pro-Vent, PulmiDur, Pulmo-Timelets, Quibron, Respid, Slo-Bid,Slo-Phyllin, Solosin, Sustaire, Talotren, Teosona, Theobid, Theoclear,Theochron, Theo-Dur, Theolair, Theon, Theophyl, Theograd, Theo-Sav,Theospan, Theostat, Theovent, T-Phyl, Unifyl, Uniphyl, Uniphyllin, andXanthium. The chemical name of Theophylline is3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione or 1,3-Dimethylxanthine andits general chemical formula is C₇H₈N₄O₂.

Aminophylline

A theophylline derivative, this is synonymous with theophyllineethylenediamine. Aminophylline is a derivative of theophylline; both aremethylxanthines and are derived from Xanthines. The drug aminophyllinediffers somewhat in its structure from theophylline in that it containsethylenediamine, as well as more molecules of water. Aminophylline tendsto be less potent and shorter acting than theophylline. Its structure isshown below:

Since a significant portion of the inhaled dose may be swallowed, it isimportant to note that theophylline is well absorbed from thegastrointestinal tract with up to 90-100 per cent bioavailability. Peaklevels are achieved within 1-2 hours following ingestion, but this isslowed by the presence of food. Theophylline is approximately 60 percent plasma protein bound and has a mean volume of distribution of0.51/kg. Plasma protein binding is reduced in infants and in patientswith liver cirrhosis. The mean plasma half-life of theophylline is about8 hours in adults although there is large intra- and inter-individualvariation, and also varies greatly with age being approximately 30 hoursin premature neonates, 12 hours within the first 6 months, 5 hours up tothe first year of life and approximately 3.5 hours up to the age of 20gradually increasing again thereafter.

Theophylline is mainly metabolised in the liver by demethylation oroxidation using the cytochrome P450 system. Only small amounts areexcreted by the kidney unchanged, and dosage adjustments in renalfailure are unnecessary. However, caution needs to be exercised whenusing other drugs that are also metabolised by the cytochrome systemwhen dosage adjustments need to be made in conjunction with themeasurement of plasma levels. Many drugs may interfere with themetabolism of theophylline. Special care should be taken with certainantibiotics as patients with acute infective exacerbations of theirairways obstruction may be inadvertently put on them withoutconsideration of the effects on theophylline metabolism. These includethe macrolide (e.g. erythromycin) and quinolone (e.g. ciprofloxacin)families of antibiotics which both reduce theophylline clearance tovarying degrees. Other drugs that reduce theophylline clearance includecimetidine, allopurinol and propanolol (although this would be a ratherunusual therapeutic combination). Drugs that increase theophyllinemetabolism include rifampicin, phenobarbitone and particularly phenytoinand carbamazepine but not the oral contraceptive pill. The rate ofmetabolism of theophylline is increased substantially in cigarettesmokers (the half life can be halved), although may not be significantin those who smoke less than 10/day. Smoking marijuana has a similareffect as can eating a high protein diet. Hepatic dysfunction, heartfailure and cor pulmonale all reduce the elimination of theophylline,and low albumin states reduce the amount of protein bound drug in theblood, so results of plasma levels need to be interpreted with caution.Therefore, as the clinical state of the patient with heart failure orrespiratory failure with cor pulmonale improves, the clearance oftheophylline alters, and dosage adjustments may be necessary.

Administering the methylxanthine by the inhaled route for pulmonarydelivery is not only desirable to maximise the locally mediatedpulmonary anti-inflammatory synergy with the inhaled steroid, but alsoto administer the methylxanthine at lower doses (<25 mg) resulting inminimal systemic exposure (<1 mg/L). Very low methylxanthine plasmalevels may significantly reduce the incidence of the systemic sideeffects of standard oral methylxanthine therapy described above.

Methylxanthines

Methylxanthine compounds, which include theophylline and aminophylline,have the general formula

Wherein

X represents hydrogen, an aliphatic hydrocarbon radical or —CO—NR₃R₄;

R₁, R₂ and R₃ represent aliphatic hydrocarbon radicals;

R₄ represents hydrogen or an aliphatic hydrocarbon radical and R₃ and R₄together with the nitrogen atom may also represent an alkylene iminoradical with 5 to 6 ring members or the morpholino radical; and

R₅ represents hydrogen or an aliphatic hydrocarbon radical.

All such compounds are within the scope of the present invention;however, theophylline itself is especially preferred.

Steroids

Steroid drugs in general are suitable for use in the present invention.Particular steroids are set forth below.

Common inhaled steroids include:

-   -   Pulmicort® (budesonide)    -   Flovent® (fluticasone)    -   Asmanex® (mometasone)    -   Alvesco® (cilcesonide)    -   Aerobid® (flunisolide)    -   Azmacort® (triamcinolone)    -   Qvar® (beclomethasone HFA)    -   Steroids may also be administered in the form of combinations        with long acting bronchodilators with a range of mechanisms        including beta 2 adrenergic agonists (such as formoterol or        salmeterol) and/or muscarinic antagonists such as ipratropium        and tiotropium. The bronchodilator included in the steroid        combination can have beta 2 adrenergic agonist and muscarinic        antagonist activity in the same molecule.    -   Advair® (Flovent® and Serevent®) Note: Serevent® is the long        acting beta-agonist salmeterol.    -   Symbicort® (Pulmicort® and Oxis®) Note: Oxis is the long acting        beta-agonist formoterol.    -   Other steroid/B2 agonist combinations are in clinical        development such as Ciclesonide and formoterol, and fluticasone        and formoterol (Flutiform). These products may also benefit from        combination therapy with inhaled theophylline.

Common steroid pills and syrups include:

-   -   Deltasone® (prednisone)    -   Medrol® (methylprednisolone)    -   Orapred®, Prelone®, Pediapred® (prednisolone)

Budesonide

Chemical name: C₂₅H₃₄O₆: 430.54 (+)-[(RS)-16a,17a-Butylidenedioxy-11b,21-dihydroxy-1,4-pregnadiene-3,20-dione]

CAS Registry Number: 51333-22-3

Budesonide was originally synthesised from 16a-hydroxyprednisolone. Theunique structure of the molecule is the key to its combination of hightopical anti-inflammatory potency with relatively low potential forsystemic side-effects. In addition, budesonide is both sufficientlywater soluble for easy dissolution in mucosal fluids and lipid solublefor rapid uptake by mucosal membranes.

Because the acetal group is asymmetrical, budesonide exists as a 1:1mixture of two epimers, known as 22R and 22S.

Fluticasone

BRAND_NAMES: Cutivate, Flixonase, Flixotide, Flonase, Flovent, Flunase

CHEMICAL_NAME:

(6(, 11 (, 16(, 17( )-6,9-difluoro-11 -hydroxy-16-methyl-3 -oxo-17-( 1-oxopropoxy)androsta-1,4-diene-17-carbothioic acid S-(fluoromethyl)ester

CHEMICAL_FORMULA: C₂₅H₃₁F₃O₅S

CAS_NUMBER: 80474-14-2

Ciclesonide

BRAND_NAMES: Alvesco

CHEMICAL_NAME:

(R)-11beta,16alpha,17,21-Tetrahydroxypregna-1,4-diene-3,20-dione, cyclic16,17-acetal with cyclohexanecarboxaldehyde, 21-isobutyrate

CHEMICAL_FORMULA: C₃₂—H₄₄—O₇

CAS_NUMBER: 141845-82-1

Beclomethasone

CHEMICAL_NAME

(11(,16()-9-chloro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione

CHEMICAL_FORMULA

C₂₂H₂₉ClO₅

CAS_NUMBER

4419-39-0

BRAND NAMES (VARIANT)

Aerobec (beclomethasone dipropionate), Aldecin (beclomethasonedipropionate), Anceron (beclomethasone dipropionate), Andion(beclomethasone dipropionate), Beclacin (beclomethasone dipropionate),Becloforte (beclomethasone dipropionate), Beclomet (beclomethasonedipropionate), Beclorhinol (beclomethasone dipropionate), Becloval(beclomethasone dipropionate), Beclovent (beclomethasone dipropionate),Becodisks (beclomethasone dipropionate), Beconase (beclomethasonedipropionate), Beconasol (beclomethasone dipropionate), Becotide(beclomethasone dipropionate), Clenil-A (beclomethasone dipropionate),Entyderma (beclomethasone dipropionate), Inalone (beclomethasonedipropionate), Korbutone (beclomethasone dipropionate), Propaderm(beclomethasone dipropionate), Qvar (beclomethasone dipropionate),Rino-Clenil (beclomethasone dipropionate), Sanasthmax (beclomethasonedipropionate), Sanasthmyl (beclomethasone dipropionate), Vancenase(beclomethasone dipropionate), Vanceril (beclomethasone dipropionate),Viarex (beclomethasone dipropionate), and Viarox (beclomethasonedipropionate).

Triamcinolone

BRAND_NAMES

Aristocort, Aristospan, Azmacort, Kenalog Nasacort

CHEMICAL_NAME

(11(,16( )-9-fluoro-11,21-dihydroxy-16,17-[1-methylethylidenebis(oxy)]pregna-1,4-diene-3,20-dione

CHEMICAL_FORMULA

C₂₄H₃₁FO₆

CAS_NUMBER

76-25-5

Salmeterol/Advair

BRAND_NAMES

*1-hydroxy-2-naphthoate *1-hydroxy-2 -naphthoate: Arial, Salmetedur,Serevent

CHEMICAL_NAME

(()-4-hydroxy-(′-[[[6-(4-phenylbutoxy)hexyl]amino]methyl]-1,3-benzenedimethanol

CHEMICAL_FORMULA

C₂₅H₃₇NO₄

CAS_NUMBER

89365-50-4

Methylprednisolone

CHEMICAL_NAME

(6(,11( )-11,17,21-trihydroxy-6-methylpregna-1,4-diene-3,20-dione

CHEMICAL_FORMULA

C₂₂H₃₀O₅

CAS_NUMBER

83-43-2

BRAND_NAMES

Medrate, Medrol, Medrone, Metastab, Metrisone, Promacortine, Suprametil,Urbason

Prednisone

BRAND_NAMES

Ancortone, Colisone, Cortancyl, Dacortin, Decortancyl, Decortin,Delcortin, Deltacortone, Deltasone, Deltison, Di-Adreson, Encorton,Meticorten, Nurison, Orasone, Paracort, Prednilonga, Pronison,Rectodelt, Sone, Ultracorten

CHEMICAL_NAME

17,21 -dihydroxypregna-1,4-diene-3,11,20,trione

CHEMICAL_FORMULA

C₂₁H₂₆O₅

CAS_NUMBER

53-03-2

Formulation

Xanthine derivatives such as theophylline and aminophylline areavailable in a variety of pharmaceutical preparations. Likewise, steroiddrugs are widely available in a variety of formulations. Formulationsused in the examples described herein are further detailed below, butany formulation may be used in the present invention which allowsinhaled delivery (oral or nasal inhalation) of the drugs to the subjectin the desired dosage.

Preferred compositions for use according to the invention may suitablytake the form of tablets, blisters, capsules, granules, spheroids,powders or liquid preparations.

In this invention theophylline is administered in inhaled form. Aerosolgeneration can be carried out, for example, by pressure-driven jetatomizers or ultrasonic atomizers, but advantageously bypropellant-driven metered aerosols or propellant-free administration ofmicronized active compounds from, for example, inhalation capsules orother “dry powder” delivery systems. Steroid drugs may be administeredby either the oral or inhaled route. Advantageously, the steroid drugsare administered by the inhaled route.

The active compounds are dosed as described depending on the inhalersystem used, in addition to the active compounds the administrationforms additionally contain the required excipients, such as, forexample, propellants (e.g. Frigen in the case of metered aerosols),surface-active substances, emulsifiers, stabilizers, preservatives,flavorings, fillers (e.g. lactose in the case of powder inhalers) or, ifappropriate, further active compounds.

For the purposes of inhalation, a large number of apparata are availablewith which aerosols of optimum particle size can be generated andadministered, using an inhalation technique which is appropriate for thepatient. In addition to the use of adaptors (spacers, expanders) andpear-shaped containers (e.g. Nebulator®, Volumatic®), and automaticdevices emitting a puffer spray (Autohaler®), for metered aerosols, inparticular in the case of powder inhalers, a number of technicalsolutions are available (e.g. Diskhaler®, Rotadisk®, Accuhaler®,Turbohaler®, Clickhaler®, Duohaler®, Evohaler®, Twisthaler®, Aerobec,Airmax, SkyeHaler, HandiHaler®, Respimat® Soft Mist™ Inhaler) or otherinhalers for example as described in European Patent Applications EP 0505 321, EP 407028, EP 650410, EP 691865 or EP 725725).

Respiratory diseases treated by the present invention specificallyinclude COPD (bronchitis, obstructive bronchitis, spastic bronchitis,emphysema), cystic fibrosis, steroid resistant asthma (including smokingasthmatics) and paediatric asthma which can be treated by thecombination according to the invention. The synergistic combination ofthe invention is particularly indicated in long-term therapy, sincelower quantities of drugs are needed than in conventional monotherapies.

1. Materials

Compounds were purchased from an external supplier.Carboxymethyl-cellulose (CMC) (Na salt) (product code C-4888) wasobtained from Sigma. Phosphate buffered saline (PBS) was obtained fromGibco. Sterile saline (0.95 w/v NaCl) and Euthatal (sodiumpentobarbitone) were obtained from Fresenius Ltd. and the VeterinaryDrug Company respectively.

The tobacco smoke was generated using 1R1 cigarettes purchased from theInstitute of Tobacco Research, University of Kentucky, USA.

Mice

Female inbred A/J mice (body weights on initial day of use: ˜20 g) wereobtained from Harlan, full barrier bred and certified free fromspecified micro-organisms on receipt. The mice were housed, up to 5 percage, in individually ventilated, polycarbonate solid bottomed cages(IVC) with grade 8 aspen chip bedding. Environment (airflow, temperatureand humidity) within the cages was controlled by the IVC system(Techniplast). Food (RM 1, Special Diet Services) and water wereprovided ad libitum. Individual animals were identified by uniquecoloured “pentel” markings on their tails, weighed and randomly assignedto treatment groups.

2. Formulation

Frequency of formulation: Compounds were formulated fresh every dayprior to each intra-nasal (i.n.) or oral dosing.

3.0 Methods

Previous studies have established that the total numbers of cellsrecovered in the BAL are significantly elevated 24 h following the finalTS exposure of 11 consecutive daily TS exposures, this time point wasused in the study reported here.

Protocols for the exposure of mice to TS, obtaining bronchoalveolarlavage (BAL), preparation of cytospin slides for differential cellcounts are as outlined below.

Exposure of Animals to TS Daily for 11 Consecutive Days

In this exposure protocol, mice were exposed in groups of 5 inindividual clear polycarbonate chambers (27 cm×16 cm×12 cm). The TS fromthe cigarettes was allowed to enter the exposure chambers at a flow rateof 100 ml/min. In order to minimise any potential problems caused byrepeated exposure to a high level of TS (6 cigarettes), the exposure ofthe mice to TS was increased gradually over the exposure period to amaximum of 6 cigarettes. The exposure schedule used in this study was asfollows:

Day 1: 2 cigarettes (approximately 16 min exposure) Day 2: 3 cigarettes(approximately 24 min exposure) Day 3: 4 cigarettes (approximately 32min exposure) Day 4: 5 cigarettes (approximately 40 min exposure) Day 5to 11: 6 cigarettes (approximately 48 min exposure)

A further group of mice were exposed to air on a daily basis forequivalent lengths of time as controls (no TS exposure).

Bronchoalveolar Lavage and Cytospin Analysis

Bronchoalveolar lavage was performed as follows:

The trachea was cannulated using a Portex nylon intravenous cannula(pink luer fitting) shortened to approximately 8 mm. Phosphate bufferedsaline (PBS) containing heparin (10 units/ml) was used as the lavagefluid. A volume of 0.4 ml was gently instilled and withdrawn 3 timesusing a 1 ml syringe and then placed in an Eppendorf tube and kept onice prior to subsequent determinations.

Cell Counts:

Lavage fluid was separated from cells by centrifugation and thesupernatant decanted and frozen for subsequent analysis. The cell pelletwas re-suspended in a known volume of PBS and total cell numberscalculated by counting a stained (Turks stain) aliquot under amicroscope using a haemocytometer.

Differential Cell Counts were Performed as Follows:

The residual cell pellet was diluted to approximately 10⁵ cells per ml.A volume of 500 μl was placed in the funnel of a cytospin slide andcentrifuged for 8 min at 800 rpm. The slide was air dried and stainedusing ‘Kwik-Diff’ solutions (Shandon) as per the proprietaryinstructions. When dried and cover-slipped, differential cells werecounted using light microscopy. Up to 400 cells were counted by unbiasedoperator using light microscopy. Cells were differentiated usingstandard morphometric techniques.

Pharmacokinetic Evaluation of Plasma Levels of Theophylline after Oralor in Dosing in TS Exposed A/J Mice

The mice were terminally anaesthetised and blood collected by cardiacpuncture into syringes containing 20 U lithium Heparin in 5 ul. Thecollected blood was mixed and decanted into eppendorf tubes beforecentrifugation in a microfuge. Plasma was collected and stored at −80° Cprior to analysis by an HPLC/MS/MS method. The equipment used in themeasurement of plasma levels were a Micromann Quatro Micro MassSpectrometer (Micromass UK Limited) and a Waters 2795 Alliance HT liquidchromatograph (Waters USA).

Six reference standard concentrations were prepared by spiking mouseplasma with stock concentrations of theophylline dissolved in methanol.Samples were prepared for analysis by adding 200 μl of acetonitrile(containing 0.25 mg/l dextrorphan as an internal standard) to 50 μl ofeach thawed sample and standard and mixed vigorously. Each sample andstandard was then centrifuged at 10000 g for 2 minutes and thesupernatant removed for LC-MS/MS analysis.

Analysis of theophylline and dextrorphan was carried out using reversephase HPLC with tandem mass spectrometric detection (LC-MS/MS). Positiveions for parent compound and a specific fragment product were monitoredin a Multiple Reaction Monitoring mode using a Micromass Quatro MicroMass Spectrometer with Micromass MassLynx software version 4.0. A 25 μlaliquot of each sample and standard was injected onto the liquidchromatography system. Lowest order of quantification (LOQ) wasdetermined as 0.016 mg/L with the detection limit being 0.003 mg/L.

3.1 Treatment Regimes

For the oral dose study, mice received vehicle (1% carboxymethylcellulose), theophylline (3 mg/kg), dexamethasone (0.3 mg/kg), atheophylline/dexamethasone combination (at 3 and 0.3 mg/kg respectively)or a theophylline/dexamethasone combination (at 1 and 0.3 mg/kgrespectively) at 1 hour prior to and 6 hours post tobacco smoke exposure(−1 h and +6 h) on each of the 11 days. In addition, animals receivingsteroid or the steroid combination were dosed with steroid 20 h prior tothe first TS exposure. Mice were additionally dosed 1 hour prior tosacrifice on the 12^(th) and final day (23 hrs post the last exposure).The control group of mice received vehicle on days 1 to 12 and wereexposed to air daily for a maximum of 50 minutes per day. BAL and plasmacollection was performed on day 12, 24 h following the eleventh andfinal TS exposure.

For the intra-nasal dose study, mice received vehicle (PBS),theophylline (0.5 mg/kg), dexamethasone (0.1 mg/kg), atheophylline/dexamethasone combination (at 0.5 and 0.1 mg/kgrespectively) a theophylline/dexamethasone combination (at 0.25 and 0.1mg/kg respectively) or a theophylline/dexamethasone combination (at 0.1and 0.1 mg/kg respectively) at 1 hour prior to tobacco smoke exposure oneach of the 11 days. In addition, animals receiving steroid or thesteroid combination were dosed with steroid 20 h prior to the first TSexposure. Mice were additionally dosed 1 hour prior to sacrifice on the12^(th) and final day (23 hrs post the last exposure). The control groupof mice received vehicle on days 1 to 12 and were exposed to air dailyfor a maximum of 50 minutes per day. BAL and plasma collection wasperformed on day 12, 24 h following the eleventh and final TS exposure.

3.2 Data Measurement and Statistical Analysis

All results are presented as individual data points for each animal andthe mean value was calculated for each group.

Since tests for normality were positive the data was subjected to a oneway analysis of variance test (ANOVA), followed by a Bonferronicorrection for multiple comparisons in order to test for significancebetween treatment groups. A “p” value of <0.05 was considered to bestatistically significant. Percentage inhibitions were automaticallycalculated within the Excel spreadsheets for the cell data using theformula below:

${\% \mspace{14mu} {Inhibition}} = {1 - {( \frac{{{Treatment}\mspace{14mu} {group}\mspace{14mu} {result}} - {{sham}\mspace{14mu} {group}\mspace{14mu} {result}}}{{{TS}\mspace{14mu} {vehicle}\mspace{14mu} {group}\mspace{14mu} {result}} - {{sham}\mspace{14mu} {group}\mspace{14mu} {result}}} ) \times 100}}$

Inhibition data for other parameters were calculated manually using theabove formula.

4.0 Results

4.1 Inflammatory Response in the Bronchoalveolar Lavage Induced byEleven Daily Consecutive Exposures to TS (24 h Post Final Exposure)

In this study exposure to TS for 11 consecutive days induced aninflammatory response 24 h following the final exposure. This consistedof significant increases in BAL of neutrophils, macrophages andlymphocytes when compared with air exposed mice (all P<0.001).

4.2 Effect of Theophylline, Dexamethasone and aTheophylline/dexamethasone Combination on the Inflammatory ResponseInduced in the Bronchoalveolar Lavage by Eleven Daily ConsecutiveExposures to TS (24 h Post Final Exposure)

Neither theophylline nor dexamethasone monotherapy when given orally orby the intra-nasal route significantly inhibited the total number ofcells recovered in the BAL induced by TS exposure (FIG. 1). Nostatistically significant inhibitory effect was seen on any of thespecific cell types (FIGS. 3-5).

In contrast, the combination of theophylline/dexamethasone at all dosesexamined whether given orally or by the intra-nasal route significantlyinhibited the total number of cells recovered in the BAL by 44-63%(p<0.001) (FIG. 1). This effect on total cells was comprised ofinhibition of macrophages, neutrophils and lymphocytes (FIG. 3-5).

A full breakdown of the inhibition of TS induced inflammatory cellinfiltrate by the combination therapy is presented in Table 1.

4.3 Pharmacokinetic Analysis

Mice received a final dose of the respective theophylline/Dexamethasonecombination therapy 1 hour prior to sacrifice and the plasma from thesemice was examined for theophylline content. Mice dosed with the oralcombination therapy had higher mean theophylline plasma content comparedwith those mice that received intra-nasal combination therapy (FIG. 2).

Oral combination therapy using 3 mg/kg or 1 mg/kg theophylline incombination with 0.3 mg/kg Dexamethasone resulted in mean theophyllineplasma levels of 1.37±0.192 mg/L and 0.479±0.047 mg/L respectively.Intra-nasal combination therapy using 0.5 mg/kg, 0.25 mg/kg or 0.1 mg/kgtheophylline in combination with 0.1 mg/kg Dexamethasone resulted inmean theophylline plasma levels of 0.074±0.013 mg/L, 0.045±0.010 mg/Land 0.022±0.009 mg/L respectively. At the doses used intra-nasaladministrated theophylline resulted in very low (<0.1 mg/L) systemictheophylline exposure 1 hour post dosing.

5.0 Discussion

In this study daily treatment with theophylline or steroid monotherapyfailed to have any inhibitory activity in this pulmonary inflammationmodel of COPD. This lack of pulmonary anti-inflammatory activity wasobserved whether the monotherapy was administered by the oral route or asurrogate route for inhalation (intra-nasal dosing). However, when thecompounds were co-administered (by either the oral or intra-nasal route)at the same doses as given alone significant anti-inflammatory activitywas demonstrated. Furthermore, lower doses of theophylline incombination with dexamethasone were also efficacious.

Moreover, the efficacy observed when the combination therapy wasadministrated by the intra-nasal route was equivalent to the efficacyobserved when the combination therapy was administrated by the oralroute despite low doses of both theophylline and dexamethasone beingused. This suggests the efficacy is being driven by local (lungspecific) anti-inflammatory activity. This [hypothesis] is furthersupported by examining the theophylline content of the plasma, asintra-nasal administration produced very low systemic levels (<0.1mg/L). As an illustration, the efficacy observed with oral combinationtherapy (1 mg/kg theophylline/0.3 mg/kg dexamethasone) on BAL totalcells (47% inhibition) was similar to that observed with intra-nasalcombination therapy (0.1 mg/kg theophylline/0.1 mg/kg dexamethasone)(44% inhibition) when the steroid was given at 3 times lower dose andtheophylline at 10 times lower dose. Furthermore, systemic theophyllinelevels were ˜17 times lower in this example. Taken together, this datasuggests that the combination therapy of theophylline and a steroidproduces a synergistic pulmonary anti-inflammatory response that ismediated in vivo specifically within the lung.

These data demonstrate the steroid insensitivity of the mouse subchronic TS model and furthermore reinforce the synergistic effect ofcombining a steroid at a therapeutic dose with an inactive dose oftheophylline as a treatment paradigm for COPD. Critically, this effectis achieved when the combination therapy is administered by a surrogatefor the inhaled route which permits very low doses of theophylline to begiven resulting in plasma levels much lower than those normallyassociated with anti-inflammatory activity (5 mg/L).

TABLE 1 Summary of the effects of theophylline, dexamethasone or theircombination on TS induced BAL inflammation observed 24 hrs following 11daily exposures BAL Inflammatory markers Total cells macrophagesneutrophils lymphocytes Treatment % Inhibition (p value) Oral dosingTheophylline (3 mg/kg) −11% (ns) −4% (ns) −10% (ns) 1% (ns)Dexamethasone (0.3 mg/kg) −16% (ns) −14% (ns) −46% (ns) −7% (ns)Theophylline (3 mg/kg + 63% (p < 0.001) 77% (p < 0.001) 66% (p < 0.05)40% (ns) Dexamethasone (0.3 mg/kg) Theophylline (1 mg/kg + 47% (p <0.001) 59% (p < 0.001) 66% (p < 0.05) 66% (p < 0.05) Dexamethasone (0.3mg/kg) Intra-nasal dosing Theophylline (0.5 mg/kg) 16% (ns) 3% (ns) 25%(ns) 14% (ns) Dexamethasone (0.1 mg/kg) 13% (ns) 10% (ns) 16% (ns) 14%(ns) Theophylline (0.5 mg/kg + 54% (p < 0.001) 60% (p < 0.001) 64% (p <0.001) 63% (p < 0.001) Dexamethasone (0.1 mg/kg) Theophylline (0.25mg/kg + 60% (p < 0.001) 59% (p < 0.001) 69% (p < 0.001) 71% (p < 0.001)Dexamethasone (0.1 mg/kg) Theophylline (0.1 mg/kg + 44% (p < 0.01) 53%(p < 0.01) 59% (p < 0.001) 69% (p < 0.001) Dexamethasone (0.1 mg/kg) ns= not statistically different from TS/vehicle group.

The in vivo studies detailed above demonstrating the synergisticanti-inflammatory activity of the pulmonary delivered combination oftheophylline and the steroid Dexamethasone using intra-nasal dosing arerepeated using other steroids including Fluticasone, Budesonide orCiclesonide. Dosages for the steroids are in the range of 1 mcg/kg/dayto 1 mg/kg/day.

It is widely accepted to those skilled in the art that doses requiredfor efficacy of inhaled drugs administered to animals can besignificantly different to those doses required for efficacy in humanindividuals. This is particularly the case for inhaled steroids.Efficacious doses of inhaled steroids administered to patients withrespiratory diseases are typically in the range of 40 mcg to 1600 mcg(total dose regardless of body weight of the individual) depending onthe steroid used and many other factors. Doses of inhaled steroidsadministered to animals are described in mass per unit body weight (e.g.mg/kg) rather than absolute dose. Converting the clinically used dosesinto mcg/kg allows comparisons between human and animal doses to becompared and demonstrates significant differences. For example,Budesonide is typically administered to patients in the range of 200mcg-1600 mcg per day, which for an average patient (70 kg) is equivalentto 2.9 mcg/kg-22.9 mcg/kg. In a mouse model of asthma, Budesonide at adose of 350 mcg/kg was efficacious but doses of 35 mcg/kg or 3.5 mcg/kgwere not when administered intra-nasally (Shen et al. Am. J. Respir.Crit Care Med. 2002, 166(2): 146-153).

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry, molecular biology and biotechnology orrelated fields are intended to be within the scope of the followingclaims.

1. A method for the treatment of chronic obstructive pulmonary disease(COPD), comprising administering by the inhaled route for pulmonarydelivery to a human subject in need thereof a composition comprisingtheophylline at a dose between 0.033 and 25 mg/day and a corticosteroidselected from the group consisting of mometasone furoate andbeclomethasone dipropionate.
 2. The method of claim 1, wherein thetheophylline is administered in an amount of 0.05 to 12.5 mg two timesper day.
 3. The method of claim 1, wherein the theophylline isadministered in an amount of 0.033 to 8 mg three times per day.
 4. Themethod of claim 1, wherein the composition comprises one or morepharmaceutically acceptable additives, diluents and/or carriers.
 5. Themethod of claim 1, wherein the theophylline is administered at a dosethat achieves plasma levels between 1 pg/L and 1 mg/L.
 6. The method ofclaim 1, wherein the inhaled corticosteroid is mometasone furoate. 7.The method of claim 6, wherein the mometasone furoate is administered atdoses between 50 μg and 1000 μg per day.
 8. The method of claim 7,wherein the mometasone furoate is administerd at daily doses between 110μg and 880 μg.
 9. The method of claim 1, wherein the inhaledcorticosteroid is beclomethasone dipropionate.
 10. The method of claim9, wherein the beclomethasone dipropionate is administered at dailydoses between 20 μg and 2000 μg.
 11. The method of claim 10, wherein thebeclomethasone dipropionate is administered at daily doses between 40 μgand to 800 μg.
 12. The method of claim 1, wherein the corticosteroid andtheophylline are co-administered.